Background: The ability to identify low-level somatic DNA mutations and minority alleles within an excess wild-type sample is becoming essential for characterizing early and posttreatment tumor status in cancer patients. Over the past 2 decades, much research has focused on improving the selectivity of PCR-based technologies for enhancing the detection of minority (mutant) alleles in clinical samples. Routine application in clinical and diagnostic settings requires that these techniques be accurate and cost-effective and require little effort to optimize, perform, and analyze. Content: Enrichment methods typically segregate by their ability to enrich for, and detect, either known or unknown mutations. Although there are several robust approaches for detecting known mutations within a high background of wild-type DNA, there are few techniques capable of enriching and detecting low-level unknown mutations. One promising development is COLD-PCR (coamplification at lower denaturation temperature), which enables enrichment of PCR amplicons containing unknown mutations at any position, such that they can be subsequently sequenced to identify the exact nucleotide change. Summary: This review summarizes technologies available for detecting minority DNA mutations, placing an emphasis on newer methods that facilitate the enrichment of unknown low-level DNA variants such that the mutation can subsequently be sequenced. The enrichment of minority alleles is imperative in clinical and diagnostic applications, especially in those related to cancer detection, and continued technology development is warranted.

Identifying low-abundance mutations within wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. However, utilizing the clinical and diagnostic potential of rare mutations is limited by sensitivity of the molecular techniques employed, especially when the type and position of mutations are unknown. We have developed a novel platform that incorporates a synthetic reference sequence within a polymerase chain reaction (PCR) reaction, designed to enhance amplification of unknown mutant sequences during COLD-PCR (CO-amplification at Lower Denaturation temperature). This new platform enables an Improved and Complete Enrichment ( ice -COLD-PCR) for all mutation types and eliminates shortcomings of previous formats of COLD-PCR. We evaluated ice -COLD-PCR enrichment in regions of TP53 in serially diluted mutant and wild-type DNA mixtures. Conventional-PCR, COLD-PCR and ice -COLD-PCR amplicons were run in parallel and sequenced to determine final mutation abundance for a range of mutations representing all possible single base changes. Amplification by ice -COLD-PCR enriched all mutation types and allowed identification of mutation abundances down to 1%, and 0.1% by Sanger sequencing or pyrosequencing, respectively, surpassing the capabilities of other forms of PCR. Ice -COLD-PCR will help elucidate the clinical significance of low-abundance mutations and our understanding of cancer origin, evolution, recurrence-risk and treatment diagnostics.

Despite recent advances in linear whole genome amplification of intact DNA/RNA, amplification of degraded nucleic acids in an unbiased fashion remains a serious challenge for genetic diagnosis. We describe a new whole genome amplification procedure, RCA–RCA (Restriction and Circularization-Aided Rolling Circle Amplification), which retains the allelic differences among degraded amplified genomes while achieving almost complete genome coverage. RCA–RCA utilizes restriction digestion and whole genome circularization to generate genomic sequences amenable to rolling circle amplification. When intact genomic DNA is used, RCA–RCA retains gene-amplification differences (twofold or higher) between complex genomes on a genome-wide scale providing highly improved concordance with unamplified material as compared with other amplification methodologies including multiple displacement amplification. Using RCA–RCA, formalin-fixed samples of modest or substantial DNA degradation were successfully amplified and screened via array-CGH or Taqman PCR that displayed retention of the principal gene amplification features of the original material. Microsatellite analysis revealed that RCA–RCA amplified genomic DNA is representative of the original material at the nucleotide level. Amplification of cDNA is successfully performed via RCA–RCA and results to unbiased gene expression analysis (R² = 0.99). The simplicity and universal applicability of RCA–RCA make it a powerful new tool for genome analysis with unique advantages over previous amplification technologies.

PCR is widely employed as the initial DNA amplification step for genetic testing and cancer biomarker detection. However, a key limitation of PCR-based methods, including real-time PCR, is the inability to selectively amplify low levels of variant alleles in a wild-type allele background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact on clinical decision-making and outcome or that can serve as cancer biomarkers. We developed COLD-PCR (co-amplification at lower denaturation temperature-PCR) [Li, Wang, Mamon, Kulke, Berbeco and Makrigiorgos (2008) Nat. Med. 14, 579–584], a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. Consequently, COLD-PCR amplification from genomic DNA yields PCR products containing high-prevalence variant alleles that can be detected. Since PCR constitutes a ubiquitous initial step for almost all genetic analysis, COLD-PCR provides a general platform to improve the sensitivity of essentially all DNA-variation detection technologies including Sanger sequencing, pyrosequencing, single molecule sequencing, mutation scanning, mutation genotyping or methylation assays. COLD-PCR combined with real-time PCR provides a new approach to boost the capabilities of existing real-time mutation detection methods. We replaced regular PCR with COLD-PCR before sequencing or real-time mutation detection assays to improve mutation detection-sensitivity by up to 100-fold and identified novel p53/Kras/EGFR (epidermal growth factor receptor) mutations in heterogeneous cancer samples that were missed by all existing methods. For clinically relevant micro-deletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR is expected to have diverse applications in the fields of biomarker identification and tracing, genomic instability, infectious diseases, DNA methylation testing and prenatal identification of fetal alleles in maternal blood.